Date of Award:

5-2005

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Science

Committee Chair(s)

Bart C. Weimer

Committee

Bart C. Weimer

Committee

Marie Walsh

Committee

Jon Takemoto

Committee

Adele Cutler

Committee

Lance Seefeldt

Abstract

Lactic acid bacteria are essential as flavor producers of cheese and fermented products. They are capable of catabolizing aromatic, branched chain, and sulfur amino acids to flavor compounds. During cheese ripening the numbers of lactococcal colonies decrease, but lactococci survive without replication in culture. This prompted an investigation into possible mechanisms of catabolism of branched chain amino acids into branched chain fatty acids and the physiological relevance of amino acid catabolism to the bacteria. We hypothesized that lactococci catabolize branched chain amino acids to branched chain fatty acids during nonculturability.

Lactococci, lactobacilli, and brevibacteria catabolized both branched chain amino acids and keto acids into branched chain fatty acids. Lactococci survived carbohydrate-limited conditions for over 4 yrs. Their survival was represented by maintaining intracellular ATP, enzyme activity, membrane integrity, capability of ATP- and PMF-dependent substrate transport, transcription, and catabolism of amino acids to fatty acids. Assays conducted with NMR spectroscopy coupled with in silico analysis showed that branched chain substrates are catabolized via keto acids, HMG-CoA, and acetyl-CoA to branched chain fatty acids. A short list of candidate genes was identified for the pathway by gene expression analysis coupled to NMR analysis. The expression of these genes and the presence of the related catabolites were identified in long-term starved cultures of nonculturable lactococci. This verified that catabolism of branched chain amino acids to branched chain fatty acids occurred during the nonculturable state only and in conditions of carbohydrate deprivation. The pathway also facilitated fixation of carbon by lactococci, revealing the mechanism of survival of lactococci over 4 yrs in culture without the addition of external carbon sources. Between strains the availability of carbohydrate and acid stress played significant roles in modulating their ability to produce branched chain catabolites.

The ability of lactococci to catabolize branched chain amino acids during sugar starvation represents a shift in carbon catabolic routes. The identified pathway also represented a balance between catabolism and anabolism, suggesting that the bacteria were in a homeostatic state during nonculturability. We accepted the hypothesis that nonculturable lactococci catabolized branched chain amino acids to branched chain fatty acids during starvation.

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